Novel thiazolinyl-picolinamide-based palladium(II) complex extenuates the virulence and biofilms of vulvovaginal candidiasis (VVC) causing Candida.

Candida infections are growing all over the world as a result of their resistance to anti-fungal drugs. This raises concerns about public health, particularly in cases of vulvovaginal candidiasis (VVC). Therefore, the need for effective treatment options for Candida infections has become crucial. The main goal of the study is to evaluate the efficacy of novel palladium metal complexes against fluconazole-resistant Candida spp., particularly C. albicans and C. auris. The process begins with identifying the minimum inhibitory concentration (MIC), followed by growth curve assays, colony morphology analysis, characterization, and gene expression analysis. The investigation revealed that sub-MIC of Pd(II) complex B (250 µg/mL) inhibited Candida spp. more effectively than amphotericin B (500 µg/mL). Further, Pd(II) complex B drastically reduced the growth of Candida spp. biofilms by 70-80% for nascent biofilms and 70-75% for mature biofilms. Additionally, the yeast-to-hyphal switch and SEM studies revealed that Pd(II) complex B effectively hinders the growth of drug-resistant Candida cells. The gene expression investigation also evidenced that Pd(II) complex B downregulated virulence genes in C. albicans (ERG, EFG, UME6, and HGC) and C. auris (ERG, CDR, and HGC). The findings showed that Pd(II) complex B effectively inhibited the growth of Candida biofilm formation and was reported as a potential anti-biofilm agent against Candida spp. that are resistant to drugs.

Cell-free supernatants of probiotic consortia impede hyphal formation and disperse biofilms of vulvovaginal candidiasis causing Candida in an ex-vivo model.

Vulvovaginal candidiasis is the second most common vaginal infection caused by drug-resistant Candida species that affects about 70-75% of reproductive age group women across the globe. As current-day antifungal drugs are ineffective against the biofilms formed by the drug-resistant Candida strains, several natural compounds and antagonistic microbes are being explored as alternative antifungal agents. In the present study, we investigated the anti-biofilm activity of Cell-Free Supernatant (CFS) extracted from the commercially available probiotics VSL-3 against the biofilms of Candida species and also evaluated their efficacy in curbing the yeast-to-hyphal transition. Various methodologies like crystal violet staining and scanning electron microscopy were used to study the effect of CFS against the biofilms formed by the species. The ability of CFS to interfere with yeast to hyphal transition in Candida was studied by colony morphology assay and visually confirmed with phase contrast microscopy. The potential of the CFS of the probiotics was also evaluated using goat buccal tissue, a novel ex-vivo model that mimics the vaginal environment. Moreover, the supernatant extracted from VSL-3 had the ability to down-regulate the expression of virulence genes of Candida from the biofilm formed over the ex-vivo model. These results emphasize the anti-fungal and anti-infective properties of the CFS of VSL-3 against drug-resistant Candida strains causing vulvovaginal candidiasis.

Development of an anti-infective urinary catheter composed of polyvinyl alcohol/sodium alginate/methylcellulose/polyethylene glycol by using a pressure-assisted 3D-printing technique.

Catheter-associated urinary tract infections (CAUTI) are a common complication associated with catheterization, leading to urosepsis, bacteriuria, and septicaemia. The present work focuses on 3D printing a urinary catheter with anti-infective properties using various concentrations of polyvinyl alcohol (PVA, e.g., 6-8 %), sodium alginate (NaAlg, e.g. 1-4 %), methylcellulose (MC, 5 %), polyethylene glycol (PEG, 5 %) impregnated with secnidazole, an antibiotic acting against Gram-negative bacteria. To produce suitable polymer ink for Pressure Assisted Microsyringe (PAM) 3D printing, the cross-linked between NaAlg and calcium chloride is necessary to prepare the catheter. The optimised catheter was found to have an outer diameter of 5 mm, an inner diameter of 3.5 mm, and a length of the catheter of 50 mm. The analysis by various methods confirms the successful incorporation of secnidazole in the 3D-printed catheter. A drug-loaded/coated catheter showed an initial drug release of 79 % following a sustained release to reach 100 % within 5 h. Weibull model fits well with the drug release data. The release models suggest the Quasi-Fickian diffusion mechanism from the system. Moreover, the secnidazole 3D printed catheter disrupted biofilms and suppressed all the Quorum sensing mediated virulence factors of two important keystone pathogens causing urinary tract infections.

Biosurfactant synergized with marine bacterial DNase disrupts polymicrobial biofilms.

Globally, the occurrence of biofilm associated infection has become an alarming menace to the medical fraternity because the thick exopolysaccharide layer encasing the biofilms makes the biofilm producing pathogens inherently resistant to antibiotics. Candida albicans, the most common pathogen among Candida spp. is the causative agent for superficial and invasive candidiasis. The morphological phase switching from yeast to hyphal form is one of the virulent traits of C. albicans critical for its pathogenicity. Owing to the emergence of antifungal resistance among this opportunistic fungus, there is a dire need for improvised alternative antifungal agents. In the present study, we have evaluated a biosurfactant from a marine bacterium for its biofilm disruption ability against C. albicans. This biosurfactant had the potential to disrupt biofilms as well as to inhibit the morphological transition from yeast to hyphae. In addition, this biosurfactant showed enhance disruption of mixed species biofilms of C. albicans and Staphylococcus epidermidis when combined with DNase isolated from marine bacteria. From the results obtained, it is evident that the biosurfactant could act as a potential antibiofilm agent against drug resistant C. albicans strains.

Chitosan-Coated Surgical Sutures Prevent Adherence and Biofilms of Mixed Microbial Communities.

Sutures are widely used materials for closing the surgical wounds, and being an inert material, sutures are often colonized with drug-resistant polymicrobial biofilms. Surgical site infection (SSI) is a hospital-acquired infection caused by bacteria and fungi specifically in the sutured sites. Although most of the currently available sutures possess antibacterial property, their ability to prevent biofilm colonization by polymicrobial communities is underexplored. So, the present study shows that extracted chitosan (EC) from crab shells prevented the adherence of Staphylococcus epidermidis and Candida albicans, the predominant members that exist as mixed species at the site of SSI. In comparison with a commercial chitosan, EC showed profound inhibition of slime formation and mixed species biofilm inhibition. Intriguingly, EC-coated sutures could inhibit the growth of both bacterial and fungal pathogens when comparing with a commercial triclosan-coated suture which was active only against the bacterial pathogen. Scanning electron microscopy results revealed inhibition of C. albicans hyphal formation by the EC-coated sutures that is a crucial virulence factor responsible for tissue invasiveness. Collectively, the results of the present study showed that EC from crab shells (discarded material as a recalcitrant biowaste) could be used as an alternative to combat drug-resistant biofilms which are the prime cause for SSIs.

Chitosan coated catheters alleviates mixed species biofilms of Staphylococcus epidermidis and Candida albicans.

Microorganisms which adhere to the surfaces of indwelling medical implants develop into a sessile microbial community to form monomicrobial or polymicrobial biofilms. Staphylococcus epidermidis and Candida albicans are the most common pathogens co-isolated from device mediated infections. Hence development of catheters coated with anti-fouling substances is of great interest. In this current study, chitosan, extracted from the shells of marine crab Portunus sanguinolentus was coated over the surface of the urinary catheters and checked for its efficacy to inhibit the adherence of both mono and mixed species biofilms. The Extracted Chitosan (EC) coated catheters showed profound activity in reducing the preformed biofilms and the other virulence factors of the pathogens like slime production in S. epidermidis and yeast to hyphal swtich in C. albicans. Furthermore, qPCR analysis showed that EC could downregulate the virulence genes in both the pathogens when grown as monospecies and mixed species biofilms.

Gallic Acid an Agricultural Byproduct Modulates the Biofilm Matrix Exopolysaccharides of the Phytopathogen Ralstonia solanacearum.

Ralstonia solanacearum is a soil-borne plant pathogen which causes wilt disease in economically important crops of the Solanaceae family in tropical and temperate regions. As biofilm formation is the major virulence factor in R. solanacearum, research inputs are necessary to identify natural biofilm inhibitors to mitigate virulence of this bacterium. Hence in the present work, the anti-biofilm potential of phytochemical compound gallic acid (GA) isolated from an agricultural byproduct (cashewnut shell) was investigated. Initially the Minimum inhibitory concentration (MIC) of crude extracts of cashewnut shell and coconut shell against R. solanacearum were investigated. The MIC of both the extracts were 400 µg/ml and their sub-MIC (200 µg/ml) inhibited biofilms in the range of 62-70% and 49-57%, respectively. As the cashewnut shell extract have higher biofilm inhibitory effect compared to coconut shell extract, we proceeded our further study by isolating the major compound GA from cashewnut shell by acid hydrolysate method. The sub-MIC of crude cashewnut shell extract inhibited 85% of young biofilms. The MIC of GA were observed at 3 mg/ml and sub-MIC (1.5 mg/ml) was found to eradicate 85% of mature biofilms which was confirmed by standard crystal violet assay and the biofilm reduction was further visualized under light microscopy and scanning electron microscopic images. Toxicity of GA was evaluated against R. solanacearum through XTT cell viability assay and found no antibacterial effect at sub-MIC. Additionally, it is confirmed with growth curve and time kill assays. Swimming and twitching motility were considered as an important virulence factors to invade plants and to block the xylem vessels. Therefore, sub-MIC of GA was found to inhibit both swimming and twitching motility of about 93% and 63% respectively. Anti-biofilm efficacy of GA was also worked well with tomato plant model where remarkable biofilm inhibition was found on treatment with GA before and after 24 h of infection with R. solanacearum. Hence GA will be an alternative, cheap source which is eco-friendly as well as novel source for the treatment of R. solanacearum biofilms and to prevent wilt disease in important crops.

Novel thiazolinyl-picolinamide based palladium(II) complex-impregnated urinary catheters quench the virulence and disintegrate the biofilms of uropathogens.

Pseudomonas aeruginosa and Serratia marcescens are prominent members belonging to the group of ESKAPE pathogens responsible for Urinary Tract Infections (UTI) and nosocomial infections. Both the pathogens regulate several virulence factors, including biofilm formation through quorum sensing (QS), an intercellular communication mechanism. The present study describes the anti-biofilm and QS quenching effect of thiazolinyl-picolinamide based palladium(II) complexes against P. aeruginosa and S. marcescens. Palladium(II) complexes showed quorum sensing inhibitory potential in inhibiting swarming motility behaviour, pyocyanin production and other QS mediated virulence factors in both P. aeruginosa and S. marcescens. In addition, the establishment of biofilms was prevented on palladium (II) coated catheters. Overall, the present study demonstrates that thiazolinyl-picolinamide based palladium (II) complexes will be a promising strategy to combat device-mediated UTI infections.

Suppressing the phenotypic virulence factors of Uropathogenic Escherichia coli using marine polysaccharide.

Uropathogenic Escherichia coli (UPEC) is one of the keystone pathogen that cause 80-90% of community acquired urinary tract infections (UTIs) and Catheter associated urinary tract infections (CAUTIs). Pathogenicity and ability of UPEC to colonize the bladder majorly relies on the expression of phenotypic virulence factors like flagella, pili, curli, and non pilus adhesion. Pathogens that colonize on the indwelling medical devices are able to communicate using quorum sensing (QS) signals. QS Plays a vital role in coordinating biofilm formation which results in the bacterial cells encased inside an extracellular polymeric substance (EPS). Chitosan is a marine polysaccharide which is known for its antibacterial activity. In the present study we investigated the ability of chitosan extracted from marine biowaste to mitigate the QS mediated biofilm formation in UPEC. Extracted chitosan (EC) and Commercial chitosan (CC) showed percentage inhibition of 80-85% and 60-75% respectively on young biofilm inhibition and preformed biofilm disruption. EC and CC were assessed for its ability to suppress QS mediated virulence in UPEC. Hemolysis assay showed a percentage inhibition of 79% against EC. Both chitosan showed profound activity to suppress the phenotypic virulence factors like swarming motility which is mediated by type I pili and colony morphology assay showed repression in cellulose production in UPEC. Furthermore, Real-Time PCR confirmed the ability of EC to down regulate the virulent genes which are responsible for invasion in UPEC. Accordingly, the current study foresees the quorum sensing inhibiting (QSI) potential of chitosan extracted from marine biowaste which offers an antibiotic free approach to combat UTI caused by UPEC.

Marine bacterial DNase curtails virulence and disrupts biofilms of Candida albicans and non-albicans Candida species.

Candida is one of the most prevalent fungal pathogens in clinical settings which form antibiotic-resistant biofilms on biomedical devices. Hence, there is a need for non-antimicrobial alternatives to combat these infections. The present study investigates the anti-biofilm effect of marine bacterial DNase by targeting the eDNA present in the biofilms of Candida spp. A strain of Vibrio alginolyticus (AMSII) which showed enhanced DNase activity was isolated from marine sediment. Treatment of young and mature Candida biofilms with purified marine bacterial DNase (MBD) caused a 60-80% reduction in biofilm biomass, similar to treatment with DNase I from Bovine pancreas. Scanning electron microscopy showed that MBD significantly reduced the formation of biofilms on urinary catheters and more importantly prevented the virulent yeast to hyphae dimorphic switch in C. albicans. The present study identified a potential non-antibiotic alternative therapy to eradicate Candida biofilms and can be used to develop enzyme fabricated antifouling indwelling medical devices.

Agro food by-products and essential oil constituents curtail virulence and biofilm of Vibrio harveyi.

Vibrio harveyi causes severe loss to the aquaculture industry due to its virulence, which is mediated by Quorum sensing (QS) and biofilm formation. In the current study, we have explored the anti-virulent properties and biofilm disruption ability of luteolin (extracted from coconut shell) and linalool against this important aquaculture pathogen. HPLC analysis of the methanolic extract of coconut shells revealed a single major peak which matched to the standard luteolin which was further elucidated by NMR studies. Further, luteolin and linalool were screened for their ability to inhibit biofilms and various quorum sensing mediated virulence factors of V. harveyi. The Minimum Inhibitory Concentration (MIC) of the two compounds was determined and the sub-inhibitory concentrations of the compounds were able to inhibit biofilm formation. Both the compounds disrupted about 60-70% mature biofilms, which was also visually observed by light microscopy. Both linalool and luteolin exhibited a significant reduction in the production of EPS and alginate in the biofilms matrix of V. harveyi which was confirmed by Scanning Electron Microscopy (SEM). Both compounds inhibited the swarming and swimming motility, the crucial quorum sensing (QS) mediated virulence of V. harveyi. The present study shows the presence of valuable polyphenolic compound like luteolin in coconut shells that are discarded as a waste. From the present study we envisage that luteolin and linalool can serve as potent anti-virulent agents to combat QS mediated infections against aquaculture pathogens.

Extracted chitosan disrupts quorum sensing mediated virulence factors in Urinary tract infection causing pathogens.

Quorum sensing (QS) plays an important role during the aetiology of urinary tract infection (UTI), as several virulence factors are under the regulation of QS. Pseudomonas aeruginosa and Serratia marcescens, the primary causative agents of UTI, employ acyl homoserine lactone (AHL) as signal molecules to coordinate various virulence factors. In this present study, chitosan extracted from the marine crab Portunus sanguinolentus was screened for its ability to inhibit the QS-signaling molecules of P. aeruginosa (PA01) and few clinical isolates of P. aeruginosa and S. marcescens. The extracted chitosan on comparison with a commercial chitosan showed significant inhibition of several QS-dependent virulence factors in P. aeruginosa and S. marscenes. Furthermore, qPCR analysis was carried out to confirm the down-regulation of fimA, fimC and flhD genes involved in adhesion and pathogenesis of S. marcescens and lasI and rhlI genes that governs the P. aeruginosa quorum sensing system. Moreover, the chitosan when coated on a catheter was also able to disrupt the mature biofilms which was revealed by scanning electron microscopy. Collectively, the present study showcases the QS inhibitory property of extracted chitosan from crab shells which is being discarded as a recalcitrant biowaste.

In vitro antibacterial activity of nut by-products against foodborne pathogens and their application in fresh-cut fruit model.

Aqueous extract of nut by-products (cashewnut shell, coconut shell, and peanut hull) were studied for their physicochemical properties, antibacterial activity and food preservation potential in an artificially inoculated fresh-cut fruit (papaya) model. Physicochemical characteristics revealed the colour, odor, nearly neutral pH (6.67-6.83), high water solubility (69.18-82.63%) and total phenolic content (1130.54-2403.41 mg GAE/100 g) of the extracts. The antibacterial property of the extracts evaluated by zone of inhibition assay revealed that cashew nut shell extract had a strong inhibition effect on Escherichia coli (18 mm), Listeria monocytogenes (18 mm), and Salmonella enterica (16 mm). Food preservative effect of extracts was examined in an artificially inoculated fresh-cut papaya model, and both cashewnut and coconut shell extracts significantly reduced the population of the above mentioned foodborne pathogens. However, when compared to coconut shell extract, the application of cashewnut shell extract was found to affect the sensory property of the fresh-cut fruit as darkening of the cut fruit was observed. So, the coconut shell extract could be considered as a natural source of antibacterial agent for food preservative applications. Phytochemical investigation through LC-MS/MS technique revealed that luteolin as the major constituent of coconut shell extract.

Use of agricultural waste (coconut shell) for the synthesis of silver nanoparticles and evaluation of their antibacterial activity against selected human pathogens.

Green synthesis of silver nanoparticles (AgNPs) is environmentally satisfactory because of their low cost and safe to nature. In the present study, extract of an agricultural waste, coconut (Cocos nucifera) shell is used to synthesize AgNPs and their antibacterial effect was investigated against selected human pathogens Staphylococcus aureus, Listeria monocytogenes, Escherichia coli, Salmonella typhimurium. The AgNPs synthesized using coconut shell extract (CSE-AgNPs) were characterized using UV-Visible spectroscopy (absorption peak at 432 nm), Transmission Electron Microscopy (spherical shaped particles size of 14.2-22.96 nm), Fourier-Transform Infrared Spectroscopy indicating the CSE capping around the AgNPs (Peaks 1384, 1609 and 3418 corresponds to organic molecules) and X-Ray Diffraction (Peak at 32.078 and 2-Theta). CSE-AgNPs exhibited zone of inhibition against S. aureus (15 mm), E. coli (13 mm), S. typhimurium (13 mm) and L. monocytogenes (10 mm) and minimum inhibitory concentration (MIC) of 26, 53, 106 and 212 µg/ml, respectively. Growth curve assay showed the effectiveness of CSE-AgNPs to inhibit the selected pathogens when compared to amphicillin control and extract. Scanning electron microscopy results indicated that the cell wall degradation might be the possible mechanism of antibacterial action of CSE-AgNPs. Different concentrations of AgNPs (0.078-2.5 mg/ml) showed no toxicity against human PBMC cell line. Hence, such highly effective CSE-AgNPs could be explored as antibacterial agent.

Essential oils from unexplored aromatic plants quench biofilm formation and virulence of Methicillin resistant Staphylococcus aureus.

In the current study we have evaluated the antibiofilm and antivirulent properties of unexplored essential oils (EOs) obtained from Pogostemon heyneanus and Cinnamomum tamala against Methicillin Resistant Staphylococcus aureus (MRSA) strains. The EOs from both the aromatic plants was screened for their ability to prevent biofilm formation and to disrupt preformed biofilms. The efficacy of both the EOs to disrupt the preformed biofilms of various MRSA strains was determined by Confocal Laser Scanning Microscopy (CLSM) and Scanning Electron Microscopy (SEM).The EOs were further able to reduce the Extracellular polymeric substance (EPS) and slime synthesis the two factors of the biofilm assemblage. The EOs was also found to be effective in reducing virulence factors like staphyloxanthin and hemolysin. In silico docking studies were performed for the major components of essential oils and dehydroxysqualene synthase of MRSA which is responsible for the synthesis of staphyloxanthin. The results suggest that (E)-nerolidol showed better binding affinity towards the enzyme. Other compounds have similar binding strengths with the enzyme. Furthermore, the synergistic effect EOs along with the commercially available DNaseI and Marine Bacterial DNase (MBD) showed that the synergistic effect had enhanced biofilm disruption ability. The results show that EOs from P. heyneanus and C. tamala has potential antivirulent and biofilm inhibitory properties against clinical and drug resistant S. aureus strains. The present study highlights the importance of bioprospecting plant based natural products as an alternative for antibiotics owing to the emergence of multi-drug resistant strains.

Chitosan extracted from marine biowaste mitigates staphyloxanthin production and biofilms of Methicillin- resistant Staphylococcus aureus.

Multidrug-resistant (MDR) Staphylococcus aureus is a major cause of biofilm-associated and indwelling device related infections. The present study explores the anti-virulent and antibiofilm potency of chitosan extracted from the shells of the marine crab Portunus sanguinolentus against Methicillin Resistant Staphylococcus aureus (MRSA). The chemical characterization results revealed that the extracted chitosan (EC) has structural analogy to that of a commercial chitosan (CC). The extracted chitosan was found to be effective in reducing the staphyloxanthin pigment, a characteristic virulence feature of MRSA that promotes resistance to reactive oxygen species. Furthermore, Confocal laser scanning microscope (CLSM) revealed that EC exhibited a phenomenal dose dependent antibiofilm efficacy against mature biofilms of the standard as well as clinical MRSA strains and Scanning Electron Microscopy (SEM) confirmed EC had a higher efficacy in disrupting the thick Exopolysaccharide (EPS) layer than CC. Additionally, EC and CC did not have any cytotoxic effects when tested on lung epithelial cell lines. Thus, the study exemplifies the anti-virulent properties of a marine bioresource which is till date discarded as a biowaste.

Jacalin-copper sulfide nanoparticles complex enhance the antibacterial activity against drug resistant bacteria via cell surface glycan recognition.

In any therapeutic modality the usage of drug in high doses often leads to serious side-effects. Herein, we demonstrated a method to enhance the antibacterial efficacy of CuS NPs at lower concentration through interacting with jackfruit seed lectin, jacalin. Fluorescence quenching studies revealed that jacalin form complex with CuS NPs and the association constant was 1.91 × 104 M-1. Upon complex with jacalin, the bacterial minimum inhibitory concentration (MIC) of CuS NPs drastically decreases from 12.5 µM to 0.78 µM. The addition of jacalin specific sugar, galactose to jacalin-CuS NPs complex (JCuS NPs) reverses the MIC from 0.78 µM to 25 µM. Mechanistic study suggests that JCuS NPs kills bacteria in part by reactive oxygen species and membrane damage, but galactose prevents the action of JCuS NPs at 0.78 µM. JCuS NPs successfully reduce (14 fold) A. hydrophila colonization in an infected zerbra fish and rescue them completely from the infection, but galJCuS NPs and CuS NPs were ineffective at 0.78 µM. Collectively, our studies demonstrates that the enhance antibacterial activity of JCuS NPs is likely due to the interaction between the galactose binding site of jacalin and the bacterial strains, as a result NPs are targeted and delivered sufficiently.

Antivirulent Properties of Underexplored Cinnamomum tamala Essential Oil and Its Synergistic Effects with DNase against Pseudomonas aeruginosa Biofilms - An In Vitro Study.

Pseudomonas aeruginosa is a nosocomial pathogen colonizing patients with chronic infectious diseases and has gained resistance to all the known broad spectrum antibiotics available today. The present study showcases the antibiofilm potential of an essential oil (EO) from an underexplored Cinnamomum species namely, C. tamala, against P. aeruginosa biofilms. Furthermore, the synergistic effects of the EO along with a commercially available DNase (DNaseI) and a DNase (MBD) isolated from a marine bacterium were explored for its antibiofilm activity. The results showed that the synergized action has maximum efficacy in inhibiting young and preformed biofilms. The synergized effect of EO and DNaseI showed 70% inhibition against matured biofilms of P. aeruginosa. The EO from C. tamala also showed quorum sensing inhibitory potential as it could inhibit the swarming motility behavior of P. aeruginosa. The synergistic action of EO and DNases offers a novel alternate therapeutic strategy for combating P. aeruginosa biofilm associated infections.

Usnic acid inhibits biofilm formation and virulent morphological traits of Candida albicans.

Biofilm formation and the yeast to hyphal switch are considered to be important virulence factors of Candida albicans. The present study reports about the potential of usnic acid, a lichen secondary metabolite inhibiting these virulent factors. Usnic acid, at its biofilm inhibitory concentration (BIC) largely reduced the viability of the metabolically active cells in matured C. albicans biofilms, exhibited significant biofilm inhibition (65%) and prevented the property of adhesion. Light microscopic images revealed that usnic acid effectively inhibited the yeast to hyphal switch and confocal microscopy showed that usnic acid greatly reduced the thickness of matured biofilms. Furthermore, usnic acid was able to reduce various sugars present in the exopolysaccharide layer (EPS) which was also confirmed by FT-IR analysis. Taken together, the present study showcases usnic acid as a potent anti-virulent compound against C. albicans and opens up a new avenue for bioprospecting lichen secondary metabolites as anti-virulent compounds.